This proposal embodies the rational design, high throughput screening, and in vitro characterization of novel neuronal actuators. The starting point for our endeavor is an ionotropic channel that launched the optogenetic revolution. We are confident that the highly original and comprehensive development scheme we have outlined will yield a new set of transformative tools for functional brain analysis. For nearly a decade, functional analysis of brain circuitry has relied on methods that allow neuronal activity to be perturbed in an intact brain with cell type-specificity. Genetically-encoded neuron actuators have ranged from chimeric G-protein coupled receptors (GPCRs) with orthogonal ligands to light-gated ionotropic channels. While these tools have helped uncover cellular substrates of cognitive and behavioral states, significant limitations remain. Optical fiber implantation is destructive, and illumination is limited by mechanical constraints and the requirement that the target site be identified in advance. GPCRs are often inefficient, display poor temporal control, and can produce long-term functional changes in neurons. We propose to develop and test a neuronal activator that embodies the strongest features of existing approaches. Based on the purinergic P2X receptor, this ionotropic channel will display high unitary conductance, negligible desensitization, and tunable gating. Its small molecule ligand will readily cross the blood-brain barrier. Complementary modifications in channel and ligand structure will help generate a family of orthogonal receptor-ligand pairs for independent control over multiple cell populations within the brain while eliminating crosstalk with endogenous factors. The strength of this and other pharmacogenetic approaches is that the locations of target neurons need not be known a priori; however, should precise temporal regulation be needed, the ligands can be chemically disabled (caged), enabling brief localized photoactivation. We are confident that our novel synthetic purinergic activator (SPArk) will advance functional brain mapping, providing robust control over discrete neuronal populations that represent known neurochemical classes or are selected using pioneering activity-based molecular-genetic methods. SPArk is a timely, highly efficient and flexible alternative to existing approaches; it is essential for continued progress in in vivo mechanistic interrogation of neuronal signaling pathways. We envision a panoply of tools that will be deployed brain-wide across species to control distinct ensembles of neurons, uncovering circuit connectivity and signaling hierarchies. However, just as with existing technologies, much work remains to be done not only to engineer the synthetic receptors, but also to synthesize and screen orthogonal ligands that are well-tolerated, easy to administer, and that readily reach target sites in the brain. We will work across experimental systems, in yeast and fibroblasts, to identify the most promising actuator candidates, to be subjected to extensive testing and optimization in vitro prior to deployment in animals.